trmD

ABSTRACT

The invention provides trmA methyl transferase (trmD) polypeptides and polynucleotides encoding trmD polypeptides and methods for producing such polypeptides by recominant techniques from strepococcus pneumoniae. Also provided are methods for utilizing polypepides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/274,825, filed Mar. 23, 1999 now U.S. Pat. No. 6,165,764. Thisapplication also claims benefit of U.S. Provisional Patent ApplicationNo. 60/107,691 filed Nov. 9, 1998.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the trmD (tRNA methyltransferases) family, as well as their variants, herein referred to as“trmD,” “trmD polynucleotide(s),” and “trmD polypeptide(s)” as the casemay be.

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Steptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcus genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Steptococcus pneumoniae strains that are resistantto some or all of the standard antibiotics. This phenonenon has createdan unmet medical need and demand for new antimicrobial agents, vaccines,drug screening methods, and diagnostic tests for this organism.

Moreover, the drug discovery process is curreny undergoing a fundamentalrevolution as it embraces “functional genomics,” that is, highthroughput genome- or gene-based biology. This approach is rapidiysoperseding earlier approaches based on “positional cloning” and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of interest from the manymolecular biology databases now available as well as from other sources.The is a continuing an significant need to identify and characterizefurther genes and other polynucleotides sequences and their relatedpolypeptides, as targets for drug discovery.

Clearly, there exists a need for polynucleotides and polypeptides, suchas the trmD embodiments of the invention, that have a present benefitof, among other things, being useful to screen compounds forantimicrobial activity. Such factors are also useful to determine theirrole in pathogenesis of infection, dysfunction and disease. There isalso a need for identification and charaction of such factors and theirantagonist and agonist to find ways to prevent, ameliorate or correctsuch infection, dysfunction and disease.

SUMMARY OF THE INVENTION

The present invention relates to trmD, in particular trmD polypeptidesand trmD polynucleotides, recombinant materials and methods for theirproduction. In another aspect, the invention relates to methods forusing such polypeptides and polynucleotides, including treatment ofmicrobial diseases, amongst others. In a further aspect, the inventionrelates to methods for identifying agonists and antagonists using thematerials provided by the invention, and for treating microbialinfections and conditions associated with such, infections with theidentified agonist or antagonist compounds. In a still further aspect,the invention relates to diagnostic assays for detecting diseasesassociated with microbial infections and conditions associated with suchinfections, such as assays for detecting trmD expression or activity.

Various changes and modifications with in the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to trmD polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a trmD of Streptococcuspneumoniae, that is related by amino acid sequence homology to B.subtilis trmD polypeptide. The invention relates especially to trmDhaving a nucleotide and amino acid sequences set out in Table 1 as SEQID NO:1 and SEQ ID NO:2 respectively. Note that sequences recited in theSequence Listing below as “DNA” represent an exemplification of theinvention, since those of ordinary skill will recognize that suchsequences can be usefully employed in polynucleotides in general,including ribopolynucleotides.

TABLE 1 trmD Polynucleotide and Polypeptide Sequences (A) Streptococcuspneumoniae trmD polynucleotide sequence [SEQ ID NO:1]. 5′-atgaagattgatattttaaccctctttccagagatgttttctccactggagcactcaatcgttggaaaggctcgagaaaaagggctcttggatatccagtatcataattttcgagaaaatgctgaaaaggcccgtcatgtagatgatgagccctacggaggcggtcagggcatgttgctcagagcacaacctattttcaattcctttgatgctattgaaaagaaaaatccgcgcgttattctcctcgatcctgctggaaagcagtttgatcaggcttatgctgaagatttggctcaagaggaagagctaatctttatctgtgggcactatgagggttatgatgagcgcattaagaccttggtaacagatgagatttccctaggcgactatgtcctcactggtggagaattggcagctatgaccatgattgatgctacagttcgcctgattccagaagtgattggcaaggagtctagccaccaagatgatagtttttcttcaggtcttttagaatatcctcagtacacacgtccctatgattatcgaggcatggtcgtgccagatgtattgatgagtggccaccatgaaaagattcgtcagtggcgattgtacgagagtttaaagaaaacctacgagcgcagaccagatttacttgaacattatcaactgacagtagaagaagaaaaaatgctggcagaaatcaaagaaaacaaagaataa -3′ (B) Streptococcus pneumoniae trmD polypeptide sequencededuced from a polynucleotide sequence in this table[SEQ ID NO:2]. NH₂-MKIDILTLFPEMFSPLEHSIVGKAREKGLLDIQYHNFRENAEVDDEPYGGGQGMLLRAQPIFNSFDAIEKKNPRVILLDPAGKQFDQAYAEDLAQEEELIFICGHYEGYDERIKTLVTDEISLGDYVLTGGELAAMTMIDATVRLIPEVIGKESSHQDDSFSSGLLEYPQYTRPYDYRGMVVPDVLMSGHHEKIRQWRLYESLKKTYERRPDLLEHYQLTVEEEKMLAEIKENKE -COOH

Deposited Materials

A deposit comprising a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Apr. 11, 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit.

On Apr. 17, 1996 a Streptococcus pneumoniae 0100993 DNA library in E.coli was similarly deposited with the NCIMB and assigned deposit number40800. The Streptococcus pneumoniae strain deposit is referred to hereinas “the deposited strain”or as “the DNA of the deposited strain.”

The deposited strain comprises a full length trmD gene. The sequence ofthe polynucleotides comprised in the deposited strain, as well as theamino acid sequence of any polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-oganism for Purposes of Patent Procedure. The deposited strainwill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strain is providedmerely as convenience to those of skill in the art and is not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strain, and compounds derived therefrom, and no suchlicense is hereby granted.

In one aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStreptococcus pneumoniae 0100993 stain, which polypeptide is comprisedin the deposited strain. Further provided by the invention are trmDpolynucleotide sequences in the deposited strain, such as DNA and RNA,and amino acid sequences encoded thereby. Also provided by the inventionare trmD polypeptide and polynucleotide sequences isolated from thedeposited strain.

Polypeptides

TrmD polypeptide of the invention is substantially phylogeneticallyrelated to other proteins of the trmD (tRNA methyl transferases) family.

In one aspect of the invention there are provided polypeptides ofStreptococcus pneumoniae referred to herein as “trmD” and “trmDpolypeptides” as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof and compositionscomprising the same.

Among the particularly preferred embodiments of the invention arevariants of trmD polypeptide encoded by naturally occurring alleles of atrmD gene.

The present invention further provides for an isolated polypeptide that:(a) comprises or consists of an amino acid sequence that has at least95% identity, most preferably at least 97-99% or exact identity, to thatof SEQ ID NO:2 over the entire length of SEQ ID NO:2; (b) a polypeptideencoded by an isolated polynucleotide comprising or consisting of apolynucleotide sequence that has at least 95% identity, even morepreferably at least 97-99% or exact identity to SEQ ID NO:1 over theentire length of SEQ ID NO:1; (c) a polypeptide encoded by an isolatedpolynucleotide comprising or consisting of a polynucleotide sequenceencoding a polypeptide that has at least 95% identity, even morepreferably at least 97-99% or exact idenitity, to the amino acidsequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.

The polypeptides of the invention included a polypeptide of Table 1 [SEQID NO:2] (in particular a mature polypeptide) as well as polypeptidesand fragments, particularly those that has a biological activity oftrmD, and also those that have a least 95% identity to a polypeptide ofTable 1 [SEQ ID NO:2] and also include portions of such polypeptideswith such portion of the polypeptide generally comprising at least 30amino acids and more preferably at least 50 amino acids.

The invention also includes a polypeptide consisting of or comprising apolypeptide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the amino terminus, X is hydrogen, a metal or any othermoiety described herein for modified polypeptides, and at the carboxylterminus, Y is hydrogen, a metal or any other moiety described hereinfor modified polypeptides, R₁ and R₃ are any amino acid residue ormodified amino acid residue, m is an integer between 1 and 1000 or zero,n is an integer 1 and 1000 or zero, and R₂ is an amino acid sequence ofthe invention, particularly an amino acid sequence selected from Table 1or modified forms thereof. In the formula above, R₂ is oriented so thatits amino terminal amino acid residue is at the left, covalently boundto R₁, and its carboxy terminal amino acid residue is at the right,covalently bound to R₃. Any stretch of amino acid residues denoted byeither R₁ or R₃, wh m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer. Otherpreferred embodiments of the invention are provided where m is aninteger between 1 and 50, 100 or 500, and n is an integer between 1 and50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived fromStreptococcus pneumoniae, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polypeptide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

A fragment is a variant polypeptide having an amino acid sequence thatis entirely the same as part but not all of any amino acid sequence ofany polypeptide of the invention. As with trmD polypeptides, may be“free standing,” or comprised within a larger polypeptide of which theyform a part or region, most preferably as a single continuous region ina single larger polypeptide.

Preferred fragments include, for example, truncation polypeptide havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, particularly a Streptococcus pneumoniae, are also preferred.Further preferred are fragments characterized by structural orfunctional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO:2, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthiesis; therefore, these variants may be employed as intermediatesfor producing the full polypeptides of the invention.

Polynucleotides

It is an object of the invention to provide polynucleotides that encodetrmD polypeptides, particularly polynucleotides that encode apolypeptide herein designated trmD.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding trmD polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO:1] that includes a full lengthgene, or a variant thereof. The Applicants believe that this full lengthgene is essential to the growth and/or survival of an organism thatpossesses it, such as Streptococcus pneumoniae.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding and/or expressing trmD polypeptides andpolnucleotides, partcularly Streptococcus pneumoniae trmD polypeptidesand polynucleotides, including, for example, unprocessed RNAs, ribozymeRNAs, mRNAs, cDNAs, genonic DNAs, B- and Z-DNAs. Further embodiment ofthe invention include biologically, diagnostically, prohylactically,clinically or therapeutically useful polynucleotides and polypeptides,and variants thereof, and compositions comprising the same.

Another aspect of the invention relates to isolated polynucleotides,including at least one full length gene, that encodes a trmD polypeptidehaving a deduced amino acid sequence of Table 1 [SEQ ID NO:2] andpolynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the invention there is atrmD polypeptide from Streptococcus pneumoniae comprising or consistingof an amino acid sequence of Table 1 [SEQ ID NO:2], or a variantthereof.

Using the invention provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1], a polynucleotide of the inventionencoding trmD polypeptide may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using Streptococcus pneumoniae 0100993 cellsas starting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as apolynucleotide sequence given in Table 1 [SEQ ID NO:1], typically alibrary of clones of chromosomal DNA of Streptococcus pneumoniae 0100993in E. coli or some other suitable host is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using stringent hybridization conditions. By sequencingthe individual clones thus identified by hybridization with sequencingprimers designed from the original polypeptide or polynucleotidesequence it is then possible to extend the polynucleotide sequence inboth directions to determine a full length gene sequence. Conveniently,such sequencing is performed, for example, using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Direct genornic DNA sequencing may also be performedto obtain a full length gene sequence. Illustrative of the invention,each polynucleotide set out in Table 1 [SEQ ID NO:1] was discovered in aDNA library derivied from Streptococcus pneumoniae 0100993.

Moreover, each DNA sequence set out in Tale 1 [SEQ ID NO:1] contains anopen reading frame encoding a protein having about the number of aminoacid residues set forth in Table 1 [SEQ ID NO:2] with a deducedmolecular weight that can be calulated using amino acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO:1, between nucleotide number 1 and the stopcodon that begins at nucleotide number 718 of SEQ ID NO:1, encodes thepolypeptide of SEQ ID NO:2.

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of: (a) a polynuclectidesequence that has at least 95% identity, even more preferably at least97, still more preferably at least 99%, yet still more preferably atleast 99.5% or exact identity to SEQ ID NO:1 over the entire length ofSEQ ID NO:1, or the entire length of that portion of SEQ ID NO:1 whichencodes SEQ ID NO:2; (b) a polynucleotide sequence encoding apolypeptide that has at least 95% identity, even more preferably atleast 97, still more preferably at least 99%, yet still more preferablyat least 99.5% or 100% exact, to the amino acid sequence of SEQ ID NO:2,over the entire length of SEQ ID NO:2.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Streptococcuspneumoniae, may be obtained by a process that comprises the steps ofscreeaing an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO:1 or a fragment thereof, andisolating a full-length gene and/or genoric clones comprising saidpolynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in Table 1 [SEQID NO:1]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecetory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also comprise at least onenon-coding sequence, including for example, but not limited to at leastone non-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of a fused polypeptide can be encoded. Incertain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof that may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 1 to the nucleotide immediatelyupstream of or including nucleotide 718 set forth in SEQ ID NO:1 ofTable 1, both of that encode a trmD polypeptide.

The invention also includes a polynucleotide consisting of or comprisinga polynucleotide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the 5′ end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, or together with Y defines a covalent bond,and at the 3′ end of the molecule, Y is hydrogen, a metal, or a modifiednucleotide residue, or together with X defines the covalent bond, eachoccurrence of R₁ and R₃ is independently any nucleic acid residue ormodified nucleic acid residue, m is an integer between 1 and 3000 orzero, n is an integer between 1 and 3000 or zero, and R₂ is a nucleicacid sequence or modified nucleic acid sequence of the invention,particularly a nucleic acid sequence selected from Table 1 or a modifiednucleic acid sequence thereof. In the polynucleotide formula above, R₂is oriented so that its 5′ end nucleic acid residue is at the left boundto R₁, and its 3′ end nucleic acid residue is at the right, bound to R₃.Any stretch of nucleic acid residues denoted by either R₁ and/or R₂,where m and/or n is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer. Where, in a preferredembodiment, X and Y together define a covalent bond, the polynucleotideof the above formula is a closed, circular polynucleotide, that can be adouble-stranded polynucleotide wherein the formula shows a first strandto which the second strand is complementary. In another preferredembodiment m and/or n is an integer between 1 and 1000. Other preferredembodiment of invention are provided where m is an integer between 1 and50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

It is most preferred that a polynucleotide of the invention is derivedfrom Streptococcus pneumoniae, however, it may preferably be obtainedfrom other organisms of the same taxonomic genus. A polynucleotide ofthe invention may also be obtained, for example, from organisms of thesame taxonomic family or order.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae trmDhaving an amino acid sequence set out in Table 1 [SEQ ID NO:2]. The termalso a polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypide (for example,polynucleotides interrupted by intergrated phage, an integratedinsertion sequence, an integrated vector sequence, an integratedtansposon sequence, or due to RNA editing or genomic DNA reorganization)together with additional regions, also may comprise coding and/ornono-coding sequences.

The invention further relates to variants of the polynucleotidesdescribe herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2]. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingtrmD variants, that have the amino acid sequece of trmD polypeptide ofTable 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, modified, deleted and/oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesand activities of trmD polypeptide.

Preferred isolated polynucleotide embodiments also includepolynucleotide fragments, such as a polynucleotide comprising a nuclicacid sequence having at least 15, 20, 30, 40, 50 or 100 contiguousnucleic acids from the polynucleotide sequence of SEQ ID NO:1, or anpolynucleotide comprising a nucleic acid sequence having at least 15,20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted fromthe 5′ and/or 3′ end of the polynucleotide sequence of SEQ ID NO:1.

Further preferred embodiments of the invention are polynucleotides thatare at least 95%, 97% or 99.5% identical over there entire length to apolynucleotide encoding trmD polypeptide having an amino acid sequenceset out in Table 1 [SEQ ID NO:2], and polynucleotides that arecomplementary to such polynucleotides. Most highly preferred arepolynucleotides that comprise a region that is at least 95% areespecially preferred. Furthermore, those at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99.5% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as a matepolypeptide encoded by a DNA of Table 1 [SEQ ID NO:1].

In accordance with certain preferred embodiments of this invention theare provided polynucleotides that hybridize, particularly under itconditions, to trmD polynucleotide sequence, such as thosepolynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. As herein used, theterms “stringent conditions” and “stringent hybridization conditions”mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash condition are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycomprising a complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said polynucleotide sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, cDNA and genonic DNA to isolatefull-length cDNAs and genomic clones encoding trmD and to isolate cDNAand genomic clones of other genes that have a high identity,particularly high sequence identity, to a trmD gene. Such probesgenerally will comprise at least 15 nucleotide residues or base pairs.Preferably, such probes will have at least 30 nucleotide residues orbase pairs and may have at least 50 nucleotide residues or base pairs.Particularly preferred probes will have at least 20 nucleotide residuesor base pairs and will have least than 30 nucleotide residues or basepairs.

A coding region of a trmD gene may be isolated by screening using a DNAsequence provided in Table 1 [SEQ ID NO:1] to synthesize anoligonucleotide probe. A labeled oligonuceotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genoic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frobman, et al., PNAS USA 85: 8998-9002, 1988).Recent modifications of the technique, exemplified by the Marathon™technology (Clonech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from mRNA extracted from a chosen tissue and an‘adaptor’ sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the “missing” 5′ end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery of treatsof and diagnostics for diseases, particularly human diseases, as furtherdiscussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonuciotides derivedfrom a sequence of Table 1 [SEQ ID NOS:1 or 2] may be used in theprocesses herein as described, but preferrably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is a mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids interior to a mature polypeptide (when amature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from a mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequence may be an inactive form of the polypeptide. Whenprosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

As will be recognized, the entire polypeptide encoded by an open readingframe is often not required for activity. Accordingly, it has becomeroutine in molecular biology to map the boundaries of the primarystructure required for activity N-treminal and C-treminal deletionexperiments. These experiments utilize exonuclease digestion or commitrestriction sites to cleave coding nucleic acid sequence. For example,Promega (Madison, Wis.) sell an Erase-a-base™ system that usesExonuclease III designed to facilitate analysis of the deletion products(protocol available at www.promega.com). The digested endpoints can berepaired (e.g., by ligation to synthetic linkers) to the extentnecessary to preserve an open reading fame. In this way, the nucleicacid of SEQ ID NO:1 readily provides contiguous fragments of SEQ ID NO:2sufficient to provide an activity, such as an enzymatic, binding orantibody-inducing activity. Nucleic acid sequences encoding suchfragments of SEQ ID NO:2 and variants thereof as described herein arewithin the invention, as are polypeptides so encoded.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, that is a precursor to a proprotein, having a leadersequence and one or more prosequences, that generally are removed duringprocessing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA costructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocess well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the preset invention, tohost cells that are genetically engineered with such expression systems,and to the production of polypeptides of the invention by recombinanttechniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al, MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electoporation,transduction, scrape loading ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, E. coli, streptomyces,cyanobacteria, Bacillus subtilis, and Streptococcus pneumoniae; fungalcells, such as cells of a yeast Kluveromyces, Saccharomyces, abasidiomycete, Candida albicans and Aspergillus; insect cells such ascells of Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,COS, HeLa C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plantcells, such as cells of a gymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among other,chromosomal-, episomal- and virus-derived vectors, for example, vetorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picornaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may comprise control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequene may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incoporated into the expressed polypeptide.These signals may be endonous to the polypeptide or they may beheterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of trmD polynucleotides andpolypeptides of the invention for use as diagnostic reagents. Detectionof trmD polynucleotides and/or polypeptides in a eukaryote, particularlya mammal, and especially a human, will provide a diagnostic method fordiagnosis of disease, staging of disease or response of an infectiousorganism to drugs. Eukaryotes, particularly mammals, and especiallyhumans, particularly those infected or suspeted to be infected with anorganism comprising the trmD gene or protein, may be detected thenucleic acid or amino acid level by a variety of well known techniquesas well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genomicDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled trmD polynucleotide sequence.Perfectly or significantly matched sequences can be distinguished fromimperfectly or more significantly mismatched duplexes by DNase or RNasedigestion, for DNA or RNA resctively, or by detecting differences inmelting temperatures or renaturation kinetics. Polynucleotide sequencedifferences may also be detected by alterations in the electrophoreticmobility of polynucleotide fragments in gels as compared to a referencesequence. This may be carried out or without denaturing agents.Polynucleotide may also detected by direct DNA or RNA sequencing. See,for example, Myers et al., Science, 230: 1242 (1985). Sequence changesat specfic locations also may be revealed by nuclease protection assays,such as RNase, V1 and S1 protection assay or a chemical cleavage method.See, for example, Cotton et al., Proc. Natl. Acad. Sci. USA, 85:4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprisingtrmD nucleotide sequence or fragments thereof can be constructed toconduct efficient screening of, for example, genetic mutations,serotype, taxonomic classification or identification. Array technologymethods are well known and have general applicability and can be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability (see, for example,Chee et al., Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostickit that comprises: (a) a polynucleotide of the present invention,preferably the nucleotide sequence of SEQ ID NO:1, or a fragmentthereof; (b) a nucleotide sequence complementary to that of (a); (c) apolypeptide of the present invention, preferably the polypeptide of SEQID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide ofthe present invention, preferably to the polypeptide of SEQ ID NO:2. Itwill be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, example, SEQ ID NO:1, that isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a detection of a stage of disease, or asusceptibility to a disease, that results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

The differences in a polynucleotide and/or polypeptide sequence betweenorganisms possessing a first phenotype and organisms possessing adifferent, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

Cells from an organism carrying mutations or polymorphisms (allelicvariatons) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScam RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR print complementary to apolynucleotide encoding trmD polypeptide can be used to identify andanalyze mutations. The invention further provides these primers 1, 2, 3or 4 nucleotides removed from the 5′ and/or the 3′ end. These primersmay be used for, among other things, amplifying trmD DNA and/or RNAisolated from a sample derived from an individual, such as a bodilymaterial. The primers may be used to amplify a polynucleotide isolatedfrom an infected individual, such that the polynucleotide may then besubject to various techniques for elucidation of the polynucleotidesequence. In this way, mutations in the polynucleotide sequence may bedetected and used to diagnose and/or prognose the infection or its stageor course, or to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more perferably infections caused byStreptococcus pneumoniae, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO:1].Increased or decreased expression of a trmD polynulceotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of trmD polypeptide compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a trmDpolypeptide, in a sample derived from a host, such as a bodily material,are well-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis,antibody sandwich assays, antibody detection and ELISA assays.

Antagonists and Agonists—Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics. See, e.g.,Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular Diseases herein mentioned. It is thereforedesirable to devise screening methods to identify compounds that agonize(e.g., stimulate) or that antagonize (e.g., inhibit) the function of thepolypeptide or polynucleotide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those that agonize or that antagonize the function of apolypeptide or polynucleotide of the invention, as well as relatedpolypeptides and polynucleotides. In general, agonists or antagonists(e.g., inhibitors) may be employed for therapeutic and prophylacticpurposes for such Diseases as herein mentioned. Compounds may beidentified from a variety of sources, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Suchagonists and antagonists so-identified may be natural or modifiedsubstrates, ligands receptors, enzymes, etc., as the case may be, oftrmD polypeptides and polynucleotides; or may be structural orfunctional mimetics thereof (see Coligan et al., Current Protocols inImmunology 1(2):Chapter 5 (1991)).

The screening methods may simply measure the binding of a candidatecompound to the polypeptide or polynucleotide, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynucleotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverseagonists, in the absence of an agonist or antagonist, by testing whetherthe candidate compound results in inhibition of activation of thepolypeptide or polynucleotide, as the case may be. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution comprising a polypeptide or polynucleotide ofthe present invention, to form a mixture, measured trmD polypeptideand/or polynucleotide activity in the mixture, and comparing the trmDpolypeptide and/or polynucleotide activity of the mixture to a standard.Fusion proteins, such as those made from Fc portion and trmDpolypeptide, as herein described, can also be used for high-throughputscreening assays to identify antagonists of the polypeptide of thepresent invention, as well as of phylogenetically and and/orfunctionally related polypeptides (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentsthat may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of sounds to identify those thatenhance (agonist) or block (antagonist) the action of trmD polypeptidesor polynucleotides, particlarly those compounds that are bacteristaticand/or bactericidal. The method of screening may involve high-throughputtechniques. For example, to screen for agonists or antagonists, asynthetic reaction mix, a cellular compartment, such as a membrane, cellenvelope or cell wall or a preparation of any thereof, comprising trmDpolypeptide and a labeled substrate or ligand of such polypeptide isincubated in the absence or the presence of a candidate molecule may bea trmD agonist or antagonist. The ablity of the candidate molecule toagonize or antagonize the trmD polypeptide is reflected in deceasedbinding of the labeled ligand or decreased production of product fromsuch substate. Molecules that bind gratuitously, i.e., without inducingthe efects of trmD polypeptide are most likely to be good antagonists.Molecules that bind well and, as the case may be, increase the rate ofproduct production from subsrate, increase signal transduction, orincrease chemical channel activity are agonists. Detection of the rateor level of, as the case may be, production of product from substrate,signal transduction, or chemical channel activity may be enhanced byusing a reporter system. Reporter systems that may be useful in thisregard include but are not limited to colorimetric, labeled substateconverted into product, a reporter gene that is responsive to changes intrmD polynucleotide or polypeptide activity, and binding assays known inthe art.

Polypeptides of the invention may be used to identify membrane bound orsoluble receptors, if any, for such polypeptide, through standardreceptor binding techniques known in the art. These techniques include,but are not limited to, ligand binding and crosslinking assays in whichthe polypeptide is labeled with a radioactive isotope (for instance,¹²⁵I), chemically modified (for instance, biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (e.g., cells, cell membranes,cell supernatants, tissue extracts, bodily materials). Other methodsinclude biophysical techniques such as surface plasmon resonance andspectroscopy. These screening methods may also be used to identifyagonist and antagonist of the polypeptide that compete with the bindingof the polypeptide to its receptor(s), if any. Standard methods forconducting such assays are well understood in the art.

The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Proteincomplexes, such as formed by trmD polypeptide associating with anothertrmD polypeptide or other polypeptide, labeled to comprise afluorescently-labeled molecule will have higher polarization values thana fluorescently labeled monomeric protein. It is preferred that thismethod be used to characterize small molecules that disrupt polypeptidecomplexes.

Fluorescence energy transfer may also be used characterize smallmolecules that intefere with the formation of trmD polypeptide dimers,trimers, tetramers or higher order structures, or structures formed bytrmD polypeptide bound to another polypeptide. TrmD polypeptide can belabeled with both a donor and acceptor fluorophore. Upon mixing of thetwo labeled species and excitation of the donor fluorophore,fluorescence energy transfer can be detected by observing fluorescenceof the acceptor. Compounds that block dimerization will inhibitfluorescence energy transfer.

Surface plasmon resonance can be used to monitor the effect of smallmolecules on trmD polypeptide self-association as well as an associationof trmD polypeptide and another polypeptide or small molecule. TrmDpolypeptide can be coupled to a sensor chip at low site density suchthat covalently bound molecules will be monomeric. Solution protein canthen passed over the trmD polypeptide-coated surface and specificbinding can be detected in real-time by monitoring the change inresonance angle caused by a change in local refractive index. Thistechnique can be used to characterize the effect of small molecules onkinetic rates and equilibrium binding constants for trmD polypeptideself-association as well as an association of trmD polypeptide andanother polypeptide or small molecule.

A scintillation proximity assay may be used to characterize theinteraction between an association of trmD polypeptide with another trmDpolypeptide or a different polypeptide. TrmD polypeptide can be coupledto a scintillation-filled bead. Addition of radio-labeled trmDpolypeptide results in binding where the radioactive source molecule isin close proximity to the scintillation fluid. Thus, signal is emittedupon trmD polypeptide binding and compounds that prevent trmDpolypeptide self-association or an association of trmD polypeptide andanother polypeptide or small molecule will diminish signal.

In other embodients of the invention there are provided methods foridentify compounds that bind to or otherwise interact with and inhibitor activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound be screened underconditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound; and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

Another example of an assay for trmD agonists is a competitive assaythat combines trmD and a potential agonist with trmD-binding molecules,recombinant trmD binding molecules, natural substrates or ligands, orsubstate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. TrmD can be labeled, such as byradioactivity or a colorimetric compound, such that the number of trmDmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

It will be readily appreciated by the skilled artisan that a polypeptideand/or polynucleotide of the present invention may also be used in amethod for the structure-based design of an agonist or antagonist of thepolypeptide and/or polynucleotide, by: (a) determining in the firstinstance the three-dimensional structure of the polypeptide and/orpolynucleotide, or complexes thereof; (b) deducing the three-dimensionalstucture for the likely reactive site(s), binding site(s) or motif(s) ofan agonist or antagonist; (c) synthesizing candidate compounds that arepredicted to bind to or react with the deduced binding site(s), reactivesite(s), and/or motif(s); and (d) tesing whether the candidate compoundsare indeed agonists or antagonists.

It wil be further appreciated that this will normally be an iterativeprocess, and this iterative process may be performed using automated andcomputer-controlled steps.

In a further a the present invention provides methods of treatingabnormal conditions such as, for instance, a Disease, related to eitheran excess of, an under-expression of, an elevated activity of, or adecreased activity of trmD polypeptide and/or polynucleotide.

If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagonist) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the trmDpolypeptide and/or polypeptide.

In still another approach, expression of the gene encoding endogenoustrmD polypeptide can be inhibited using expression blocking techniques.This blocking may be targeted against any step in gene expression, butis preferably targeted against transcription and/or tanslation. Anexamples of a known technique of this sort involve the use of antisensesequences, either internally generated or separately administered (see,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides thatform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgrain negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extacellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial trmD proteins that mediate tissue damage and/or; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

In accordance with yet another aspect of the invention, there areprovided trmD agonists and antagonists, preferably bacteristic orbactericidal agonists and antagonists.

The antagonist and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

Helicobacter pylori (herein “H. pylori”) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, clasifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists oftrmD polypeptides and/or polynucleotides) found using screens providedby the invention, or known in the art, particularly narrow-spectrumantibiotics, should be useful in the treatmet of H. pylori infection.Such treatment should decrease the advent of H. pylori-induced cancers,such as gastrointestinal carcinoma. Such treatment should also prevent,inhibit and/or cure gastric ulcers and gastritis.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicted tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

GLOSSARY

The following definitions are provided to facilitate understaning ofcertain terms used frequently herein.

“Bodily material(s) means any material derived from an individual orfrom an organism infecting, infesting or inhabiting an individual,including but not noted to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semens, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials.

“Disease(s)” means any disease caused by or related to infection by abacteria, including, for example, otitis media, conjuctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid.

“Host cell(s)” is a cell that has been introduced (e.g., transformed ortransfected) or is capable of introduction (e.g., transformation ortransfection) by an exogenous polynucleotide sequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Compuational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SLAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1)387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul. S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computr Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a95, 97, 99.5 or 100% identity to the reference sequence of SEQ ID NO:1,wherein said polynucleotide sequence may be identical to the referencesequence of SEQ ID NO:1 or may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO:1 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO:1, or:

n_(n)≦x_(n)−(x_(n)•y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.95 for 95%, 0.97 for97%, 0.995 for 99.5% or 1.00 for 100%, and • is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in his coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 95, 97 or 100% identity to apolypeptide reference sequence of SEQ ID NO:2, wherein said polypeptidesequence may be identical to the reference sequence of SEQ ID NO:2 ormay include up to a certain integer number of amino acid alterations ascompared to the reference sequence, wherein said alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at tha amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of amino acid alterations is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of amino acids in SEQ ID NO:2, or:

n_(a)≦x_(a)−(x_(a)•y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and • is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

“Individual(s)” means a multicellular eukaryote, including, but notlimited to a metzoan, a mammal an ovid, a bovid, a simian, a primate,and a human.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide a is introduced intoan organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Organism(s)” mmeans a (i) pro karyote icluding but not limited to, amember of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neissera, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further induding, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aerusinosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii and Chlamydia trachomitis, (n) an archaeon,including but not limited to Archaebacter, and (iii) a unicellular orfilamentous eukaryote, including but not limited to, a protozoan, afungus, a member of the genus Saccharomyces, Kluveromyces, or Candida,and a menber of the species Saccharomyces ceriviseae, Kluveromyceslactis, or Candida albicans.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single anddouble-straned regions, hybrid molecules comprising DNA and RNA that maybe single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Theso in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that comprise one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great vatiety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skilled in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein compising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may comprise amimo acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may comprise many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent of a nucleotide or nucleotidederivative, covalent attachment of a lipid or lipid derivative, covalentattachment of phosphotidylinositol, cross-linking, cyclization,disulfide bond formation, demethylation, formation of covalentcross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,iodination, methlation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,tansfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton W. H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular peptides may resultfrom post-translational natural processes and may be made by entirelysynthetic methods, as well.

“Recombinant expression system(s)” refers to expression systems orportions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substuitions, whereby a residueis substituted by another with like characteristics. Typical suchsubstitutions are among Ala, Val, Leu, and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresides Lys and Arg; or aromatic residues Phe and Tyr. Particularlypreferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 aminoacids are substituted, deleted, or added in any combination. A variantof a polynucleotide or polypeptide may be a naturally occurring such asan allelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques, by direct synthesis,and by other recombinant methods known to skilled artisans.

EXAMPLES

The examples below are carried out using standard techniques, that arewell known and routine to those of skilled in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain Selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1]was obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two ormore clones comprising overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO:1. Librariesmay be prepared by routine methods, for example:

Methods 1 and 2 Below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2

TrmD Characterization

The S. pneumoniae trmD gene is expressed during infection in arespiratory tract infection model

The determination of expression during infection of a gene fromStreptococcus pneumoniae Excised lungs from a 48 hour respiratory tractinfection of Streptococcus pneumoniae 0100993 in the mouse isefficiently disrupted and processed in the presence of chaotropic agentsand RNAase inhibitor to provide a mixture of animal and bacterial RNA.The optimal conditions for disruption and processing to give stablepreparations and high yields of bacterial RNA are followod by the use ofhybridisation to a radiolabelled oligonucleotide specific toStreptococcus pneumoniae 16S RNA on Northern blots. The RNAase free,DNAase free, DNA and protein free preparations of RNA obtained aresuitable for Reverse Transcription PCR (RT-PCR) using unique primerpairs designed from the sequence of each gene of Streptococcuspneumoniae 0100993.

a) Isolation of tissue infected with Streptococcus pneumoniae 0100993from a mouse animal model of infection (lungs)

Streptococcus pneumoniae 0100993 is seeded onto TSA (Tryptic Soy Agar,BBL) plates containing 5% horse blood and allowed to grow overnight at37° C. in a CO2 incubator. Bacterial growth is scraped into 5 ml ofphosphate-buffered saline (PBS) and adjusted to an A600˜0.6 (4×106/ml).Mice (male CBA/J-1 mice, approximately 20 g) were anaesthetized withisoflurane and 50 microliters of the prepared bacterial inoculum isdelivered by intranasal instillation. Animals are allowed to recover andobserved twice daily for signs of moribundancy. Forty-eight hours afterinfection the animals are euthanized by carbon dioxide overdose andtheir torsos swabbed with ethanol and then RNAZap. The torso is thenopened, and the lungs are aseptically removed. Half of each pair oflungs is placed in a cryovial and immediately frozen in liquid nitrogen;the other half is used for bacterial enumeration after homogenization ofthe tissue in 1 ml of PBS.

b) Isolation of Streptococcus pneumoniae 0100993 RNA from infectedtissue samples

Infected tissue samples, in 2-ml cryo-strorage tubes, are removed from−80° C. storage into a dry ice ethanol bath. In a microbiological safetycabinet the samples are disrupted up to eight at a time while theremaining samples are kept frozen in the dry ice ethanol bath. Todisrupt the bacteria within the tissue sample, 50-100 mg of the tissueis transfered to a FastRNA tube containing a silica/ceramic matrix(BIO101). Immediately, 1 ml of extraction reagents (FastRNA reagents,BIO101) are added to give a sample to reagent volume ratio ofapproximately 1 to 20. The tubes are shaken in a reciprocating shaker(FastPrep FP120, BIO101) at 6000 rpm for 20-120 sec. The crude RNApreparation is extracted with chloroform/isoamyl alcohol, andprecipitated with DEPC-treated/Isopropanol Precipitation Solution(BIO101). RNA preparations are stored in this isopropanol solution at−80° C. if necessary. The RNA is pelleted (12,000 g for 10 min.), washedwith 75% ethanol (v/v in DEPC-treated water), air died for 5-10 min, andresupended in 0.1 ml of DEPC-treated water, followed by 5-10 minutes at55° C. Finally, after at least 1 minute on ice, 200 units of Rnasin(Promega) is added.

RNA preparations are stored at −80° C. for up to one month. For longerterm storage the RNA precipitate can be stored at the wash stage of theprotocol in 75% ethanol for at least one year at −20° C.

Quality of the RNA isolated is assessed by running samples on 1% agarosegels. 1×TBE gels stained with ethidium bromide are used to visualisetotal RNA yields. To demonstrate the isolation of bacterial RNA from theinfected tissue 1×MOPS, 2.2M formaldehyde gels are run and vacuumblotted to Hybond-N (Amersham). The blot is then hybridised with a32P-labelled oligonucleotide probe, of sequence 5′AACTGAGACTGGCTTTAAGAGATTA 3′ [SEQ ID NO:3], specific to 16S rRNA ofStreptococcus pneumoniae. The size of the hybridising band is comparedto that of control RNA isolated from in vitro grown Streptococcuspneumoniae 0100993 in the Northern blot. Correct sized bacterial 16SrRNA bands can be detected in total RNA samples which show degradationof the mammalian RNA when visualised on TBE Sels.

c) The removal of DNA from Streptococcus pneumoniae-derived RNA

DNA was removed from 50 microgram samples of RNA by a 30 minutetreatment at 37° C. with 20 units of RNAase-free DNAaseI (GenHunter) inthe buffer supplied in a final volume of 57 microliters.

The DNAase was inactivated and removed by treatment with TRIzol LSReagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol.

DNAase teated RNA was resuspended in 100 microliters of DEPC treatedwater with the addition of Rnasin as described before.

d) The preparation of cDNA from RNA samples derived from infected tissue

3 microgram samples of DNAase treated RNA are reverse transcribed usinga SuperScript Preamplification System for First Strand cDNA Synthesiskit (Gibco BRL, Life Technologies) according to the manufacturersinstructions. 150 nanogram of random hexamers is used to prime eachreaction. Controls without the addition of SuperScriptII reversetranscriptase are also run. Both +/−RT samples are treated with RNaseHbefore proceeding to the PCR reaction

e) The use of PCR to determine the presence of a bacterial cDNA species

PCR reactions are set up on ice in 0.2 ml tubes by adding the followingcomponents: 43 microliters PCR Master Mix (Advanced BiotechnologiesLtd.); 1 microliter PCR primers (optimally 18-25 basepairs in length anddesigned to possess similar annealing temperatures), each primer at 10mM initial concentration; and 5 microliters cDNA.

PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 asfollows: 2 minutes at 94° C., then 50 cycles of 30 seconds each at 94°C., 50° C. and 72° C. followed by 7 minutes at 72° C. and then a holdtemperature of 20° C. (the number of cycles is optimally 30-50 todetermine the appearance or lack of a PCR product and optimally 8-30cycles if an estimation of the starting quantity of cDNA from the RTreaction is to be made); 10 microliter aliquots are then run out on 1%1×TBE gels stained with ethidium bromide, with PCR product, if present,sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5′endwith a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantiy detectedusing a suitable gel scanning system (e.g. ABI Prism™ 377 Sequencerusing GeneScan™ software as supplied by Perkin Elmer).

RT/PCR controls may include +/− reverse transcriptase reactions, 16SrRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Streptococcus pneumoniae 0100993 genomicsequences.

To test the efficiency of the primer pairs they are used in DNA PCR withStreptococus pneumoniae 0100993 total DNA. PCR reactions are set up andrun as described above using approx. 1 microgram of DNA in place of thecDNA.

Primer pairs which fail to give the predicted sized product in eitherDNA PCR or RT/PCR are PCR failures and as such are uninformative. Ofthose which give the correct size product with DNA PCR two classes aredistinguished in RT/PCR: 1. Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and 2. Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stonger signal in the +RT samples than the signal (if at allpresent) in −RT controls.

Example 3

The trmD gene is essential for S. pneumoniae in vitro growth.

Demonstration of gene essential to bacterial viability

An allelic replacement cassette was generated using PCR technology. Thecassette consisted of a pair of 500 bp chromosomal DNA fragmentsflanking an erythromycin resistance gene. The chromosomal DNA sequencesare the 500 bp preceding and following the DNA sequence encoding thetrmD gene contained in Seq. ID NO.1.

The allelic replacement cassette was introduced into S. pneumoniae R6 bytransformation. Competent cells were prepared according to publishedprotocols. DNA was introduced into the cells by incubation of ngquantities of allelic replacement cassette with 10⁶ cells at 30° C. for30 minutes. The cells were transferred to 37° C. for 90 minutes to allowexpression of the erythromycin resistance gene. Cells were plated inagar containing lug erythromycin per ml. Following incubation at 37° C.for 36 hours, colonies are picked and grown overnight in Todd-Hewittbroth supplemented with 0.5% yeast extract. Typically 1000 transformantscontaining the appropriate allelic replacement are obtained. If notransformants are obtained in three separate transformation experimentsas was the case for this gene trmD, then the gene is considered as beingessential in vitro.

3 1 720 DNA Streptococcus pneumoniae 1 atgaagattg atattttaac cctctttccagagatgtttt ctccactgga gcactcaatc 60 gttggaaagg ctcgagaaaa agggctcttggatatccagt atcataattt tcgagaaaat 120 gctgaaaagg cccgtcatgt agatgatgagccctacggag gcggtcaggg catgttgctc 180 agagcacaac ctattttcaa ttcctttgatgctattgaaa agaaaaatcc gcgcgttatt 240 ctcctcgatc ctgctggaaa gcagtttgatcaggcttatg ctgaagattt ggctcaagag 300 gaagagctaa tctttatctg tgggcactatgagggttatg atgagcgcat taagaccttg 360 gtaacagatg agatttccct aggcgactatgtcctcactg gtggagaatt ggcagctatg 420 accatgattg atgctacagt tcgcctgattccagaagtga ttggcaagga gtctagccac 480 caagatgata gtttttcttc aggtcttttagaatatcctc agtacacacg tccctatgat 540 tatcgaggca tggtcgtgcc agatgtattgatgagtggcc accatgaaaa gattcgtcag 600 tggcgattgt acgagagttt aaagaaaacctacgagcgca gaccagattt acttgaacat 660 tatcaactga cagtagaaga agaaaaaatgctggcagaaa tcaaagaaaa caaagaataa 720 2 239 PRT Streptococcus pneumoniae2 Met Lys Ile Asp Ile Leu Thr Leu Phe Pro Glu Met Phe Ser Pro Leu 1 5 1015 Glu His Ser Ile Val Gly Lys Ala Arg Glu Lys Gly Leu Leu Asp Ile 20 2530 Gln Tyr His Asn Phe Arg Glu Asn Ala Glu Lys Ala Arg His Val Asp 35 4045 Asp Glu Pro Tyr Gly Gly Gly Gln Gly Met Leu Leu Arg Ala Gln Pro 50 5560 Ile Phe Asn Ser Phe Asp Ala Ile Glu Lys Lys Asn Pro Arg Val Ile 65 7075 80 Leu Leu Asp Pro Ala Gly Lys Gln Phe Asp Gln Ala Tyr Ala Glu Asp 8590 95 Leu Ala Gln Glu Glu Glu Leu Ile Phe Ile Cys Gly His Tyr Glu Gly100 105 110 Tyr Asp Glu Arg Ile Lys Thr Leu Val Thr Asp Glu Ile Ser LeuGly 115 120 125 Asp Tyr Val Leu Thr Gly Gly Glu Leu Ala Ala Met Thr MetIle Asp 130 135 140 Ala Thr Val Arg Leu Ile Pro Glu Val Ile Gly Lys GluSer Ser His 145 150 155 160 Gln Asp Asp Ser Phe Ser Ser Gly Leu Leu GluTyr Pro Gln Tyr Thr 165 170 175 Arg Pro Tyr Asp Tyr Arg Gly Met Val ValPro Asp Val Leu Met Ser 180 185 190 Gly His His Glu Lys Ile Arg Gln TrpArg Leu Tyr Glu Ser Leu Lys 195 200 205 Lys Thr Tyr Glu Arg Arg Pro AspLeu Leu Glu His Tyr Gln Leu Thr 210 215 220 Val Glu Glu Glu Lys Met LeuAla Glu Ile Lys Glu Asn Lys Glu 225 230 235 3 25 DNA Streptococcuspneumoniae 3 aactgagact ggctttaaga gatta 25

What is claimed is:
 1. An isolated polypeptide comprising SEQ ID NO:2.2. A composition comprising the isolated polypeptide of claim 1 and acarrier.
 3. The isolated polypeptide of claim 1, wherein the isolatedpolypeptide comprises a heterologous amino acid sequence fused to SEQ IDNO:2.
 4. A composition comprising the isolated polypeptide of claim 1and a carrier.
 5. The isolated polypeptide of claim 1, wherein theisolated polypeptide consists of SEQ ID NO:2.
 6. A compositioncomprising the isolated polypeptide of claim 5 and a carrier.